NADH-linked fumarate reductase and NADH dehydrogenase activities inFibrobacter succinogenes

Crude membrane preparation fromFibrobacter succinogenes S85 were investigated and found to contain NADH dehydrogenase (NADH:decylubiquinone oxidoreductase) and NADH-linked fumarate reductase activities. Under aerobic conditions the maximum NADH dehydrogenase activity (252 nmoles/min/mg protein) was ten times greater than that of NADH-fumarate reductase (23 nmoles/min/mg protein). NADH-fumarate reductase was strongly inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), rotenone, HgCl₂, ando-phenanthroline. Inhibition of the NADH dehydrogenase by the first three compounds, particularly rotenone, accounted for most of the effects on NADH-fumarate reductase. The -band of ab-type cytochrome was resolved into two cytochromes, a cytochromeb ₅₆₀ (oxidized by addition of HOQNO) and a cytochromeb ₅₆₃ (oxidized by subsequent addition of fumarate).Published with the approval of the Director of the Agricultural Experiment Station, North Dakota State University, as journal article no. 2137.     

The dependence on quinone specificity of terminal electron transport of bacteria

Bacterial quinones were extracted with pentane, and homologues or other quinones were reincorporated. In spite of the redox potential difference of 110 mV, menaquinone and demethylmenaquinone could replace each other in aerobic electron transport and fumarate respiration ofHaemophilus influenzae RAMC 18 Bensted andProteus mirabilis Harding & Nicholson. The enzymes involved may recognize the naphthoquinone structure and are not specific for menaquinone or demethylmenaquinone. Ubiquinone was not replaced in aerobic electron transport by naphthoquinones withPseudomonas fluorescens 28/5 Rhodes orAcinetobacter sp. 661/60 Mannheim, probably owing to the specificity for benzoquinones of the enzymes involved, since the redox potential difference between demethylmenaquinone and ubiquinone is only 76 mV.Haemophilus parainfluenzae 429 Pittman, which resembles aerobic bacteria with respect to the terminal electron transport system, could incorporate demethylmenaquinone or menaquinone. This organism seems to be defective in the synthesis of naphthoquinones but possesses the enzyme system for fumarate respiration.Haemophilus influenzae RAMC 18 Bensted, which produces only demethylmenaquinone, seems to be defective in synthesizing ubiquinone, but it also possesses the enzymes for a ubiquinonemediated aerobic respiration.     

Function of fumarate reductase in methanogenic bacteria (Methanobacterium)

Methanogenic bacteria contain high activities of fumarate reductase. An interesting hypothesis has recently been advanced that this enzyme, in cooperation with a succinate dehydrogenase, functions in a fumarate-succinate cycle for ATP synthesis. This hypothesis was tested by determining whether [2, 3-³H] succinate loses³H when taken up by growing cells.Methanobacterium thermoautotrophicum was grown on H₂ plus CO₂ in the presence of [U-¹⁴C, 2,3-³H] succinate. The double labelled dicarboxylic acid was found to be incorporated into cell material with the loss of only 30% of tritium. Neither was³H released into H₂O in significant amounts. This finding excludes a catabolic oxidation of succinate to fumarate in the growing cells and thus the operation of a fumaratesuccinate cycle. It is shown that the function of fumarate reductase inM. thermoautotrophicum is to provide the cells with succinate for the synthesis of -ketoglutarate, an intermediate in glutamate, arginine and proline synthesis.     

Purification of secreted α-amylases by immunoaffinity chromatography with cross-reactive antibody

Two isozymes of rice -amylases expressed and secreted by recombinant yeast were purified by immunoaffinity chromatography by using cross-reactive antibody. Antibodies raised against partially purified barley -amylase adsorbed rice -amylases in fermentation broth by a cross-reaction. By use of these antibodies as ligands, rice -amylases were concentrated and purified to a high degree in one-step immunoaffinity chromatography. Because of the differences in the contaminating impurities between the barley -amylase (antigen) from barley malt and rice -amylases (target protein) secreted from yeast, the high purity of eluted -amylases was attained without the use of highly purified antigen for immunization. Utilization of cross-reactive antibodies in immunoaffinity chromatography is useful for the purification of recombinant proteins in the absence of a sufficient amount and high enough purity of the target proteins to be purified.     

Relationships among isolates of oral haemophili as determined by DNA-DNA hybridization

In order to assess the relationships among strains of the genera Actinobacillus and Haemophilus, DNAs from 50 strains of these genera were isolated and purified. The guanine plus cytosine (G+C) content of DNAs from strains of Haemophilus segnis and Haemophilus para-influenzae were determined by thermal denaturation. DNA-DNA homologies were measured using labelled probes from one strain representing Haemophilus segnis (strain ATCC 10977), and two strains representing Haemophilus parainfluenzae (strains ATCC 9796 and ATCC 7901). Strains isolated as H. segnis had a G+C content of 39.0 to 42.9% and were 49–92% homologous with the ATCC 10977 DNA probe. All of the strains freshly isolated as H. parainfluenzae were 70–81% homologous with the ATCC 9796 DNA probe and had a G+C content of 34.9 to 38.3%. Strain ATCC 7901 was 11% homologous with the ATCC 9796 DNA probe, had a G+C content of 42.4%, and was 65–78% homologous to DNA from strains identified as Haemophilus aphrophilus and Haemophilus paraphrophilus. From these results we conclude that strain ATCC 7901 is a mislabelled strain of H. paraphrophilus. The results of multiple DNA-DNA hybridizations indicated that separate species designations were appropriate for H. segnis, H. parainfluenzae, Actinobacillus actinomycetemcomitans (Haemophilus actinomycetemcomitans), and H. aphrophilus. H. aphrophilus and H. paraphrophilus were closely related organisms and did not fulfill the generally accepted criteria for designation as separate species.     

Intermediate metabolism inTrypanosoma cruzi

Epimastigotes ofTrypanosoma cruzi, the causative agent of Chagas disease, catabolize proteins and amino acids with production of NH₃, and glucose with production of reduced catabolites, chiefly succinate andl-alanine, even under aerobic conditions. This aerobic fermentation of glucose is probably due to both the presence of low levels of some cytochromes, causing a relative inefficiency of the respiratory chain for NADH reoxidation during active glucose catabolism, and the lack of NADH dehydrogenase and phosphorylation site I, resulting in the entry of reduction equivalents into the chain mostly as succinate. Phosphoenol pyruvate carboxykinase and pyruvate kinase may play an essential role in diverting glucose carbon to succinate orl-alanine, andl-malate seems to be the major metabolite for the transport of glucose carbon and reduction equivalents between glycosome and mitochondrion. The parasite contains proteinase and peptidase activities. The major lysosomal cysteine proteinase, cruzipain, has been characterized in considerable detail, and might be involved in the host/parasite relationship, in addition to its obvious role in parasite nutrition. Among the enzymes of amino acid catabolism, two glutamate dehydrogenases (one NADP- and the other NAD-linked), alanine aminotransferase, and the major enzymes of aromatic amino acid catabolism (tyrosine aminotransferase and aromatic -hydroxy acid dehydrogenase), have been characterized and proposed to be involved in the reoxidation of glycolytic NADH.     

Photochemical activities and photosynthetic ATP formation in membrane preparation from a facultative methylotroph,Protaminobacter ruber strain NR-1

Membrane preparation from the bacteriochlorophyll-containing cells of a facultative methylotroph, Protaminobacter ruber strain NR-1, contained reaction center bacteriochlorophyll similar to those in many species of purple bacteria and contained a few cytochrome species. -Peak of the reduced-minus-oxidized difference spectrum of one of the cytochromes was at 554 nm. The midpoint potential of the cytochrome at pH 7 (E_m7) was 350 mV. Two other cytochromes had the same reduced-minus-oxidized difference spectra with a split -band at 557 and 566 nm, but had two different E_m7s' of 130 mV and 0 mV.On flash or continuous light the reaction center bacteriochlorophyll and the cytochrome with -peak at 554 nm were reversibly oxidized. Redox titration of the light-induced cytochrome oxidation gave an E_m7 value of 356 mV. Under continuous illumination the membrane preparation reversibly took up protons, and formed ATP in the presence of ADP and inorganic phosphate. The ATP formation activity on the bacteriochlorophyll basis was one-third to one-fifth that in chromatophores from Rhodospirillum rubrum under similar experimental conditions. These results clearly indicated that the membrane preparation from P. ruber which contained bacteriochlorophyll had a cyclic photosynthetic electron transfer system and coupled ATP formation activity.Abbreviations Bchl (only in figure legends) bacteriochlorophyll - CCCP carbonylcyanide-m-chlorophenylhydrazone - E_h the ambient redox potential - E_m7 the midpoint potential at pH 7 - PMS N-methylphenazonium methosulfate - MES morpholinoethanesulfonic acid - MOPS morpholinopropanesulfonic acid     

Anaerobic metabolism of goldfish,Carassius auratus (L.). Influence of anoxia on mass-action ratios of transaminase reactions and levels of ammonia and succinate

Summary Changes in the concentrations of ammonia, glutamate, alanine, aspartate, -ketoglutarate, oxaloacetate and succinate were measured in freeze-clamped lateralred muscle, dorsal white muscle and liver, and in rapidly cooled blood of goldfish after 12 h of anoxia. Alanine accumulation, succinate accumulation and aspartate depletion are observed in all tissues examined; in the liver the concentrations of glutamate increase and those of ammonia decrease. The mass-action ratio of the glutamate-pyruvate transaminase-catalyzed reaction stays within one order of magnitude from thermodynamic equilibrium in the direction of alanine formation. The mass-action ratio of the glutamate-oxaloacetate transaminase reaction is far from equilibrium when measured oxaloacetate concentrations are used. When levels of free oxaloacetate are calculated from LDH and MDH equilibrium constants, the mass-action ratio of glutamate-oxaloacetate transamination is close to equilibrium in the direction of aspartate formation. Since neither alanine nor glutamate decreases, and since ammonia gradients suggest a continuous ammonia production in all tissues examined, anaerobic proteolysis is assumed. A possible coupling between amino acid catabolism and ethanol production is discussed.Abbreviations ALA alanine - ASP aspartate - EDTA ethylene diamine tetraacetate - FP _ox oxidated flavoprotein - FP _red reduced flavoprotein - FUM fumarate - GLU glutamate - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase - IMP inosine monophosphate - KG -ketoglutarate - LDH lactate dehydrogenase - MAL malate - MAR mass action ratio - MDH malate dehydrogenase - OAA oxaloacetate - PYR pyruvate - sAMP adenylosuccinate - SDH succinate dehydrogenase - SUCC succinate     

Evolution of chlorocatechol catabolic pathways

The aerobic bacterial degradation of chloroaromatic compounds often involves chlorosubstituted catechols as central intermediates. They are converted to 3-oxoadipate in a series of reactions similar to that for catechol catabolism and therefore designated as modifiedortho-cleavage pathway. Among the enzymes of this catabolic route, the chlorocatechol 1,2-dioxygenases are known to have a relaxed substrate specificity. In contrast, several chloromuconate cycloisomerases are more specific, and the dienelactone hydrolases of chlorocatechol catabolic pathways do not even convert the corresponding intermediate of catechol degradation, 3-oxoadipate enol-lactone. While the sequences of chlorocatechol 1,2-dioxygenases and chloromuconate cycloisomerases are very similar to those of catechol 1,2-dioxygenases and muconate cycloisomerases, respectively, the relationship between dienelactone hydrolases and 3-oxoadipate enol-lactone hydrolases is more distant. They seem to share an / hydrolase fold, but the sequences comprising the fold are quite dissimilar. Therefore, for chlorocatechol catabolism, dienelactone hydrolases might have been recruited from some other, preexisting pathway. Their relationship to dienelactone (hydrolases identified in 4-fluorobenzoate utilizing strains ofAlcaligenes andBurkholderia (Pseudomonas) cepacia is investigated). Sequence evidence suggests that the chlorocatechol catabolic operons of the plasmids pJP4, pAC27, and pP51 have been derived from a common precursor. The latter seems to have evolved for the purpose of halocatechol catabolism, and may be considerably older than the chemical industry.     

Anaerobic reduction of fumarate in the body wall musculature ofArenicola marina (Polychaeta)

Summary The reduction of fumarate, which is a characteristic feature of anoxic catabolism of some invertebrates, was investigated in mitochondria or mitochondrial fragments prepared from the body wall musculature of the lug-wormArenicola marina (Annelida, Polychaeta).A coupling of the reduction of fumarate to succinate to the oxidation of NADH was demonstrated.The pathway of hydrogen transfer from NADH to fumarate was studied by using specific inhibitors of the respiratory chain. From the results it is concluded that parts of the respiratory chain are involved.During anaerobiosis mitochondria formed succinate at a high rate from malate which had been added as substrate. The formation of succinate is coupled to oxidative phosphorylation. The ratio ATP-production/formation of succinate was found to be 0.6 to 0.8.Succinate formation from malate is inhibited by arsenite and monofluoroacetate.TheK _m for fumarate of the fumarate reductase inArenicola body wall musculature is 2.5×10^–5 M.Abbreviations Ap₅A P¹,P⁵-di(adenosine-5-)pentaphosphate - APAD acetylpyridine adenine dinucleotide - DNP 2,4-dinitrophenol - fw fresh weight (of body wall musculature) - NaFAc sodiummonofluoroacetic acid - PCA perchloric acid - PEP phosphoenolpyruvate Supported by Deutsche Forschungsgemeinschaft (Ze 40/13, Ze 40/14 and Gr 456/5)     

Low temperatures stabilize interferon α-2 against proteolysis in Methylophilus methylotrophus and Escherichia coli

Summary The accumulation of interferon (IFN) -2 in transformed strains of Escherichia coli and Methylophilus methylotrophus was greater at 25° C than at 37° C. Interferon -2 catabolism was followed by measuring the change in IFN titre (measured immunoreactively) with time at temperatures between 25° C and 37° C in chloramphenicol-treated cells. The IFN -2 titre remained constant at 29° C and below, while at higher temperatures the titres declined. The t _1/2 values for IFN -2 decreased with increasing incubation temperature. Pulse-chase studies using [³⁵S]methionine, sodium dodecyl sulphate-gel electrophoresis and autoradiography demonstrated that IFN -2 was subjected to degradation at 37° C while at 25° C it was stable. It is proposed that the susceptibility of IFN -2 to degradation in both E. coli and M. methylotrophus is affected by incubation temperature and 30° C may be a transition temperature above which the conformation of the molecule is recognised by the bacterial proteases.     

Chromosomal loci of genes controlling site-specific restriction endonucleases of Bacillus subtilis

Summary In Saccharomyces cerevisiae, diploid strains which are respiratory deficient (e.g., rho^–) or are homozygous for the mating-type locus (i.e., either a/a or /) are unable to sporulate. In order to induce sporulation in these nonsporulating strains, the technique of protoplast fusion mediated by polyethylene glycol was adopted. In this study, the products of protoplast fusion were induced to sporulate without reversion to normal cells.Protoplasts from a respiratory-deficient diploid strain were mixed with those from a respiratory-competent haploid one carrying mitochondrial drug resistance markers, treated with 30% polyethylene glycol-4000 and 25 mM CaCl₂, and incubated in 0.1 M potassium acetate containing 0.8 M sorbitol as an osmotic stabilizer. After two days' incubation, asci with three to eight spores were formed at a frequency of 1×10^–3 to 2×10^–4. Sporulation was also observed in products of fusion between an a/a diploid and haploid strains and between an / diploid and a haploid strains. The analysis of the genotypes of spores revealed that when fusion products were cultured under conditions for sporulation, karyogamy did not take place, diploid nuclei underwent meiosis, and both diploid and haploid nuclei were able to develop into spores.     

Anaerobic respiration of fumarate as a differential test betweenCampylobacter fetus andCampylobacter jejuni

The effect of formate and of various electron acceptors—fumarate, aspartate, or nitrate—on the growth of 36 catalase-producingCampylobacter strains was quantitatively investigated in a semisynthetic medium, under aerobic (5% oxygen, 10% carbon dioxide, 85% nitrogen) or anaerobic (10% carbon dioxide, 90% nitrogen) conditions. Under anaerobic conditions, 24 strains ofC. jejuni failed to grow, or grew poorly, in the presence of fumarate, whereas ten strains ofC. fetus subsp.fetus and two strains ofC. fetus subsp.venerealis grew abundantly, rather better than under aerobic conditions. The quantitative differences of growth yields were very significant between the two species with fumarate, but less pronounced with aspartate or nitrate. The activity of fumarate-reductase inC. fetus was substantiated by identification of relevant metabolites by gas liquid chromatography and by mass spectrometry. The anaerobic fumarate respiration in the genusCampylobacter has taxonomic implications.     

Biochemical and genetic evidence of benzylsuccinate synthase in toluene-degrading,ferric iron-reducing Geobacter metallireducens

In vitro assays demonstrated that toluene-grown cells of Geobacter metallireducens catalyzed the addition of toluene to fumarate to form benzylsuccinate under anaerobic conditions. The specific in vitro rate of benzylsuccinate formationwas ca. 45% of the specific in vivo rate of toluene consumption. In addition, bssA and bssB, which code for the and subunits of benzylsuccinate synthase (BSS), respectively, were found to have sequences in G. etallireducens similar to the only sequences heretofore available (for three denitrifying strains). This is the first report of the presence of BSS in a ferriciron-reducing bacterium; BSS activity has previously been reported in denitrifying, sulfate-reducing, and anoxygenic phototrophic toluene degraders, as well as in a highly enriched methanogenic, toluene-degrading culture.     

Oxidation of sulfur compounds and electron transport in Thiobacillus denitrificans

Summary Intact cells of Thiobacillus denitrificans catalyzed the oxidation of thiosulfate, sulfide and sulfite with nitrate or oxygen as the terminal acceptor. The anaerobic oxidation of thiosulfate, sulfide and sulfite was sensitive to the inhibitors of the flavoprotein system. Under aerobic conditions the oxidation of sulfide and sulfite was sensitive to these inhibitors but the thiosulfate oxidation was unaffected. Cyanide and azide inhibited the aerobic and anaerobic respiration when thiosulfate, sulfide or sulfite served as electron donors. The oxidation of thiosulfate by cell-free preparations was mediated by cytochromes of c, a and o-types. The cell-free extracts also catalyzed the oxidation of NADH and succinate, involving flavoproteins and b, c, a and o-type cytochromes. In addition, a cytochrome oxidase sensitive to cyanide and azide was also present.Non-Standard Abbreviations TTFA Thenoyltrifluoroacetone - HQNO 2-heptyl-4-hydroxyquonoline N-oxide Aspirant van het Nationaal Fonds voor Wetenschappelijk Onderzoek (Belgian National Science Foundation).     

Phorphorylative electron transport chains lacking a cytochromebc 1 complex

Electron transport-coupled phosphorylation with fumarate as terminal acceptor inWolinella succinogenes yields less than 1 ATP/2 electrons. The generated by the electron transport is 0.18V and the H⁺/electron ratio is 1. The electron transport chain is made up of two dehydrogenases (hydrogenase and formate dehydrogenase) that catalyze the reduction of menaquinone, and fumarate reductase which catalyzes the oxidation of menaquinol.C-type cytochromes are not involved. The phosphorylative electron transport with sulfur as terminal acceptor inW. succinogenes orDesulfuromonas acetoxidans does not involve known quinones. The ATP yields should be even smaller than those with fumarate. Succinate oxidation by sulfur, which is a catabolic reaction inD. acetoxidans, is accomplished by reversed electron transport.     

Regulation of intracellular H+ balance in Zymomonas mobilis 113 during the shift from anaerobic to aerobic conditions

Summary The energetics, enzyme activities and end-product synthesis of Zymomonas mobilis 113 in continuous culture were studied after the shift from an anaerobic to an aerobic environment. Aeration diminished ethanol yield and lactic acid concentration, but increased glucose consumption rate and production of acetic acid. After the shift to aerobic conditions reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]-oxidase activity was stimulated. Washed cell suspensions consumed oxygen with glucose, lactate and ethanol as substrates. The aerobic Z. mobilis 113 regulated their intracellular redox balance by production and reoxidation of the end products, coupled with the formation of NAD(P)H. An increase in transmembrane pH gradient (pH) and a decrease in intracellular ATP concentration were observed after the shift to aerobic conditions. At low medium redox potential (Eh) values the H⁺ balance was regulated in an energy-independent way via end-product excretion. Under aerobic conditions this was supplemented by ATP-dependent H⁺ excretion by the membrane H⁺-ATPase.Abbreviations D dilution rate (h^-1) - S ₀ initial glucose concentration (g/l) - Y _x/s growth yield (g/mol) - Y _p/s product yield (g/g) - q _s specific rate of substrate utilization (g/g per hour) - q _p specific rate of ethanol formation (g/g per hour) - qo ₂ specific rate of CO₂ production (mmol/g per hour) - specific growth rate (h^-1) - X dry biomass concentration (g/l) - Eh redox potential of culture medium (mV) - pH transmembrane pH gradient (pH units) - pH_in intracellular pH - SASE sum of activities of specific enmymes of Entner-Doudoroff pathway     

Alpha-1 and alpha-2 adrenoceptor binding in cerebral cortex: Role of disulfide and sulfhydryl groups

The triated adrenergic antagonists Prazosin ([³H]PRZ) and Idazoxan ([³H]IDA, or RX-781094) bind specifically and with high affinity to ₁ and ₂-adrenoceptors respectively, in membrane preparations from cerebral cortex. Saturation experiments performed to determine the density of receptors and the dissociation constant (K _d) were analyzed by the methods of Eadie Hofstee, iterative modelling, and the procedure of Hill, while the specificity of the labelling was verified by displacement experiments. Since receptors are proteins, we examined the role of disulfide (–SS–) bridges and sulfhydryl (–SH) groups in the specific combination of [³H]PRZ and [³H]IDA to the ₁ and ₂-adrenoceptors. Pretreatment of the membranes with the –SS– reactive DL-dithiothreitol (DTT) or the alkylating agent N-ethylmaleimide (NEM), alone or in combination, decreased specific binding of both ligands, with only minor changes in the non-specific counts. The [³H]IDA binding (₂-sites) was more sensitive to both DTT and NEM than the [³H]PRZ sites (₂-adrenoceptors), and the initial changes induced by alkylation of the ₂-site were due to an important decrease in the affinity for [³H]IDA, as judged by the increase in theK _d. This modulation in the affinity caused by alkylation of a thiol group could explain the higher potency of the blocking agent tetramine disulfide benextramine at the ₂-site. The results provide evidence for the participation of –SS– and –SH groups in the binding site of ₁ and ₂-adrenoceptors in the cerebral cortex.     

Dual Effect of Isoprostanes on the Release of [<Superscript>3</Superscript>H]D-Aspartate from Isolated Bovine Retinae: Role of Arachidonic Acid Metabolites

The effect of 8-isoprostanes on potassium (K⁺)-depolarization-evoked release of [³H]D-aspartate from bovine isolated retinae was investigated. Isolated bovine retinae were prepared for studies of K⁺-evoked release of [³H]D-aspartate using the Superfusion Method. Low concentrations of 8-isoPGF₂(1–100 nM) inhibited whereas higher concentrations of this 8-isoprostane (100 nM–30 M) enhanced K⁺-induced [³H]D-aspartate overflow. The excitatory effect of 8-isoPGF₂ was mimicked by thromboxane receptor agonist, U-46619 and blocked by thromboxane receptor antagonist, SQ 29,548 (10 M). Pretreatment of tissues with the cyclooxygenase (COX) inhibitor, flurbiprofen unmasked an inhibitory effect of high concentrations of 8-isoPGF₂(1–30 M) on [³H]D-aspartate release that was attenuated by AH 6809 (10 M). In conclusion, 8-isoPGF₂ exhibits a dual regulatory effect on K⁺-induced [³H]D-aspartate release in isolated bovine retinae. The inhibitory action caused by 8-isoPGF ₂ is due to the activation of EP₁/EP₂ receptors while the excitatory effects are due to the activation of thromboxane receptors.